1. Field of the Invention
The present invention is in the field of conducting an assay of a sample containing an analyte of interest.
2. Description of the Background Art
In recent years there has been increased interest in the synthesis, characterization and application of metal-ligand complexes in biomolecule research. In addition to their use as photosensitizers, metal-ligand complexes have been used as luminescence probes in polymers. For instance, metal-containing intercalators such as square-planar platinum(II) complexes containing aromatic terpyridine or phenanthroline ligands have been used in probing DNA structure and the intercalation process itself. The reagent methidiumpropyl-Fe(II) EDTA, which contains a redox-active metal center tethered to an organic intercalator, has been applied in "footprinting" experiments to determine the sequence specificity of small drugs bound to DNA. Ru(II) and Os(II) transition metal compounds have been used to probe DNA structure and study long-range electron transfer.
More recent studies have shown that ruthenium (Ru(II)), rhenium (Re(I)) and osmium (Os(II)) metal-ligand complexes display high anisotropy in the absence of rotational diffusion. Importantly, metal ligand complexes display luminescence decay times ranging from 100 ns to 100 .mu.s. Consequently, these probes extend the observable timescale of anisotropy decay measurements by orders of magnitude compared with that observable with routinely used organic fluorophores. As a result of this, metal-ligand complexes have been used to probe the microsecond dynamics of DNA. In addition, time-resolved anisotropy measurements of proteins can be extended to the microsecond timescale using metal-ligand complexes. Intensity and anisotropy decays of Ru(II) metal-ligand complexes when covalently linked to human serum albumin, concanavalin A, human immunoglobulin G and ferritin demonstrated that this class of probes could be used to measure rotational motions from 10 ns to 1.5 .mu.s, which so far has been inaccessible using the classical organic fluorophores. Fluorescence polarization immunoassays using metal-ligand complexes covalently bound to human serum albumin (as the antigen) demonstrated the potential use of metal-ligand complexes in fluorescence polarization immunoassays of high-molecular-weight analytes.
Fluorescence polarization (FP) was first theoretically described by Perrin in 1926, which was subsequently expanded and measured by Weber. Dandliker and co-workers adapted FP for use in analytical biochemistry including antigen (Ag)-antibody (Ab) interactions, and hormone-receptor interactions. Since establishment of the theory and method by Dandliker, the use of FPI's (fluorescence polarization immunoassays) for the quantitative and qualitative measurement of various types of molecules and bioconjugates has been reported. These include therapeutic drug monitoring, determination of hormones, drugs of abuse, proteins and peptides, proteases and inhibitors, as well as DNA binding interactions. In fact, FPI technology is presently in widespread commercial use in several instruments.
A serious limitation of present immunoassays is that they are limited to low molecular weight antigens. This limitation is a result of the use of fluorophores, such as fluorescein, which display lifetimes near 4 ns. A FPI requires that emission from the unbound labeled antigen be depolarized, so that an increase in polarization may be observed upon antigen binding to antibody. For depolarization to occur, the antigen must display a rotational correlation time much shorter than the lifetime of the probe (in the case of fluorescein, less than 4 ns) which limits the dynamic range of the FPI to antigens with low molecular weights (FIG. 16). Some long lifetime fluorophores, such as chelates of Eu.sup.3+ and Tb.sup.3+ have been used in time-resolved immunoassays, but they do not display polarized emission and are thus not useful in FPI's.
More recent studies have shown that [Ru(bpy).sub.2 (dcb)].sup.2+, where bpy is 2,2'-bipyridine and dcb is 4,4'-dicarboxylic acid-2,2'-bipyridine, displays high polarization in the absence of rotational diffusion (.about.0.25), as well as a long lifetime (.about.400 ns). The experimental results demonstrated that the steady-state polarization of [Ru(bpy).sub.2 (dcb)].sup.2+ labeled to HSA was sensitive to the binding of anti-HSA, which resulted in a 200% increase in polarization. Another metal-ligand complex, [Os(bpy).sub.2 (dcb)].sup.2+, was also used in a FPI to detect a high molecular weight bioconjugate using red excitation and emission wavelengths.
Many different approaches have been used to circumvent the present limitation of FPI's to low molecular weight substances. An early attempt to develop FPI's for high molecular weight antigens was reported by Grossman. The dansyl (dimethylaminonaphthalene sulfonic acid) fluorophore was used because of its lifetime near 20 ns. Tsuruoka and coworkers attempted to develop a FPI with IgG by increasing the molecular weight of the antibody. This was accomplished by immobilizing the antibody with latex or colloidal silver. Urios and Cittanova decreased the size of the labeled antibody by using Fab fragments in place of complete IgG molecules. Another approach to enable the measurement of high-molecular-weight antigens was introduced by Wei and Herron. They used a tetramethylrhodamine-labeled synthetic peptide, which has a high binding affinity for the Ab of hCG (human chorionic gonadotrophin), as the tracer antigen in their FPI for hCG. In the assay, the tracer antigen, which has a low molecular weight, is replaced by hCG (high molecular weight) thus reducing the amount of polarization.
Since the basic theory of the depolarization of fluorescence through Brownian rotation was presented by Perrin in 1926, fluorescence anisotropy decay measurements have been widely used to study the rotational dynamics of proteins, membrane-bound proteins and other macromolecules. The use of the polarization of extrinsic fluorescent labels to study proteins was introduced by Weber and was applied to the characterization of a number of proteins by Weber and others.
There are limitations imposed by the short fluorescence lifetime that have been circumvented by use of phosphorescence anisotropy decays, which have been used to study the rotational dynamics of membrane-bound proteins. Such measurements are based exclusively on the triplet probe eosin, which displays a millisecond phosphorescence decay time in the absence of oxygen. Rotational motions have been quantified by transient absorption anisotropy and by time-resolved phosphorescence anisotropy. There are, however, relatively few useful triplet probes. The use of phosphorescence is also inconvenient because of the need to rigorously exclude molecular oxygen, and the low initial phosphorescence anisotropies, typically 0.1 or smaller.
The polarized luminescence from metal ligand complexes has been used to study macromolecular dynamics. Studies have shown that ruthenium (Ru), rhenium (Re) and osmium (Os) metal ligand complexes display high anisotropy in the absence of rotational diffusion. Importantly, these metal ligand complexes display luminescence decay times ranging from 100 ns to 100 .mu.s. Consequently, these probes extend the observable timescale of anisotropy decay measurements by many-fold compared with that observable with routinely used fluorophores. As a result, metal ligand complexes have been used to measure rotational motion of proteins and probe the submicrosecond dynamics of DNA. Time-resolved anisotropy measurements have demonstrated that metal ligand complexes when covalently linked to human serum albumin (HSA), concanavalin A, human immunoglobin G, and ferritin can be used to measure rotational motions on the 10 ns to 1.5 .mu.s timescale.
Conventional organic fluorophores typically have a lifetime in the range of 1-10 ns, generally absorb in the high energy range, and are not very photostable. These properties limit the ability of these fluorophores to study slower domain-to-domain motions in proteins or the rotational motions of membrane-bound proteins. Furthermore the sensitivity of these fluorophores is also limited by interfering autofluorescence which also occurs at the 1-10 ns time scale.
There remains a need in the art for metal-ligand complex probes which display long absorption emission wavelengths, long lifetimes, high luminescence, and/or high quantum yields, for use as biomolecular probes, and/or for metal-ligand complex probes that can be used in fluorescence polarization immunoassays of high molecular weight analytes, and for metal-ligand complex probes that can be used as anisotropy probes for protein hydrodynamics.